A new validated facile HPLC analysis method to determine methylprednisolone including its derivatives and practical application

Methylprednisolone sodium succinate (MPSS) is a parenteral water-soluble corticosteroid ester. It gives three peaks methylprednisolone (MP), 17-methylprednisolone hemisuccinate (17-MPHS), and methylprednisolone hemisuccinate (MPHS) that share in the assay determination as total MP. It is used on a wide scale in prescribed anti-inflammatory drugs as a common use. The current study aimed to find a rapid RP-HPLC method of MP and its derivatives analysis with high linearity, repeatability, sensitivity, selectivity, and inexpensive to use without the need for any special chemical reagents. The use of the current method achieved a satisfactory result to detect, determine and separate the MP, 17-MPHS, and MPHS in a short time. The chromatographic system consists of RP-HPLC using the BDS column (250 mm × 4.6 mm × 5 μm). The mobile phase was prepared by mixing the WFI: glacial acetic acid: acetonitrile in a volume ratio (63:2:35) at a flow rate of 2.0 mL/min with detection wavelength at 254 nm at room temperature and injection volume 20 μL. The method manifested a satisfied linearity regression R2 (0.9998–0.99999) with LOD 143.97 ng/mL and 4.49 µg/mL; and LOQ 436.27 ng/mL and 13.61 µg/mL for MP and MPHS respectively. The method proved its efficiency via system suitability achievement in the robustness and ruggedness conduction according to the validation guidelines. High sensitivity according to its LOD and LOQ. So, the current method could be considered in the pharmaceutical industry. The suggested method has been successfully implemented in the Egyptian local market for the quantitative assessment of the assay of the finished product.

www.nature.com/scientificreports/ analysis method functions under simple chemical conditions and is easily accessible to any general laboratory. An analytical comparison of the determination of methylprednisolone employing various methods was also done.

Materials and methods
Methyl Prednisolone reference standard (MP), Methyl Prednisolone hemisuccinate reference standard (MPHS), and Methylprednisolone sodium succinate working standard (MPSS) was supplied by UP pharma (Assuit, Egypt). Acetonitrile HPLC-grade, disodium hydrogen phosphate, sodium dihydrogen phosphate, glacial acetic acid 99%, hydrochloric acid 37%, sodium hydroxide, and Hydrogen peroxide 30% (Scharlau, Spain). Water for injection (WFI) was used in the analysis and passed through a 0.45 μm nylon membrane filter before use. Phosphate solution (1) was prepared by weighing 1.6 g of disodium hydrogen phosphate in 1000 mL of WFI. Phosphate solution (2) was prepared by weighing about 0.3 g of sodium dihydrogen phosphate in 1000 mL of WFI.
Chromatographic system configuration. Compared with the previously conducted HPLC methods and the current analysis method, we did not use a high percentage of the organic modifier of acetonitrile, dedicated pH solution adjustment, or special chemical reagent to realize the optimum separation for the ideal system suitability achievement. MP, 17-MPHS, and MPHS assay determination were conducted using the HPLC model HP 1100 series with variable wavelength. The current method was conducted with the RP-BDS column (250 mm × 4.6 mm × 5 μm) (Thermo Scientific). The mobile phase was prepared as WFI: glacial acetic acid: acetonitrile in a volume ratio (63:2:35) at a flow rate of 2.0 mL/min with detection wavelength at 254 nm at room temperature and injection volume 20 μL.
Parameters of method validation. The HPLC validation method was performed according to the International Conference on Harmonization (ICH) guidelines concerning parameters including system suitability, Range of linearity, the limit of detection (LOD), the limit of quantification (LOQ), repeatability (precision), recovery and accuracy, robustness, ruggedness, the stability of the solution, specificity, and selectivity 20-22 . Sample preparations. System suitability check. System suitability was performed by injecting six replicate injections of the same sample solution which was prepared by dissolving a quantity of MP reference standard equivalent to 5 mg/100 mL of mobile phase and mixing 10 mL of this solution with a weight of MPSS working standard equivalent to 65 mg and 1 mL of each phosphate buffer solutions in 100 mL volumetric flask and complete with mobile phase to obtained a concentration about 500 µg/mL of total MP.
Range and linearity. The analytical approach is deemed to be linear if there is a substantial portion between the response and claimed working concentration starting at the lowest point in the tested range and increasing to the highest point with R 2 ≥ 0.999 [22][23][24][25][26][27] .
Regression linearity equation: where (Y) represents the response of the average peak area, (X) represents the claimed working concentration in (%), (a) represents the slope and (b) is the intercept of the calibration curve. The linearity parameter was submitted using different five concentrations in the range (50-150%) of the MP working standard. The stock solutions were prepared as a quantity of MP reference standard 48.9 mg in 100 mL of the mobile phase and complete with the WFI to 1000 mL and MPSS working standard equivalent to 640 mg/100 mL in the mobile phase. Then make serial dilutions to obtain concentrations (50%, 70%, 100%, 120%, and 150%) by taking (5 mL, 7 mL, 10 mL, 12 mL, and 15 mL) from each solution of the stock solutions and complete to 100 mL with mobile phase and inject 2 replicates of each concentration.
Limit of detection (LOD). It was defined as the lowest specified analyte concentration in the matrix that could be identified using the detection of the instrument. LOD concentration should not undergo the accuracy, precision, and linearity ranges every time it is injected [22][23][24][25][26][27] .
Limit of quantitation (LOQ). It was defined as the lowest specified analyte concentration in the matrix that could be identified using the detection of the instrument. LOQ must undergo the accuracy, precision, and linearity ranges every time it is injected [22][23][24][25][26][27] .
LOD and LOQ could be calculated according to the slope and standard error data from the linearity of the calibration as the following: where (σ) is the standard error of (X & Y) arrays and (S) represents the slope of the linearity calibration curve.
Accuracy and recovery. Both recovery and accuracy are used alternatively 28 . The measurement's accuracy is defined as the proximity of the actual concentration (measured value) to the theoretical concentration (true value) 18,20,29 . www.nature.com/scientificreports/ Accuracy was implemented by the preparation of three different stock solutions of MP reference standard at 3.74, 5.49, and 6.64 mg in 100 mL mobile phase individually. Then 10 mL of each 45.7 mg, 64.8 mg, 77.4 mg/100 mL WFI of MPSS working standard individually respectively and 1 mL of each phosphate buffer solution were mixed with MP concentrations. Then injected three replicates of each concentration were to make 70%, 100%, and 120% concentrations of total MP. Accuracy % could be estimated using the linearity equation: Repeatability and precision. Repeatability was conducted using six different determinations of the 100% test concentration by dissolving about a quantity of MP reference standard equivalent to 5 mg/100 mL of mobile phase and mixing 10 mL of this solution with a weight of MPSS working standard equivalent to 65 mg and 1 mL of each phosphate buffer solutions in 100 mL volumetric flask and complete with mobile phase to obtained a concentration about 500 µg/mL of total MP 22,30 .
Robustness. Robustness was submitted using designed small changes including slight changes in the temperature, composition of the mobile phase, etc. 22 . The designed small changes were conducted in a different organic solvent ratio (Acetonitrile) at (± 1%) and a flow rate (± 0.005 mL/min).
Ruggedness. Ruggedness was submitted using designed and major observable changes including analyst-analyst, column-column, and day-day with maintaining all of the analysis method parameters and conditions as it is without changes 23 .
Specificity and selectivity. The following solutions were injected individually for selectivity confirmation:

Test of the validated method of the local market product of UP Pharma in Egypt.
Methylprednisolone 1.0 g vials batch number (221160) after the constitution stability studies. The after-constitution stability study was conducted using the supplied solvent WFI at zero time, 24 h in the refrigerator at a temperature of 5 ± 3 °C. The constituted vial was performed using 16 mL of the WFI then all of the content of the vial was transferred into a 200 mL volumetric flask. Then a dilution of 10 mL of the constituted solution (1 mg/mL) in a 100 mL volumetric flask using WFI was conducted and introduced to the HPLC for assay in a final theoretical concentration (0.5 mg/mL of MP).
Experimental work and methods. I confirm that all methods were carried out following relevant guidelines and regulations.

Results and discussions
System suitability check. According to the molecular data in Table 1, the first eluted is MP according to its lower molar mass. Subsequently, 17-MPHS will elute then MPHS according to their stereochemistry where 17-MPHS has a smaller stereo shape as manifested in Fig. 1. So, the MP, 17-MPHS, and MPHS peaks appeared at retention times 4.7, 5.3, and 9.0 ± 0.2 min at the optimum parameters of the analysis method as shown in Fig. 2. The range of retention time over all the parameter changes for the three successive peaks were (4.7, 5.3, and 9.0) ± 1 min. Accuracy and recovery. Tables 5 and 6 showed that the accuracy results of the tested range (70-120% from the target concentration of 100% = 500 µg/mL) were found to be within the acceptance criteria (98-102%) 20 .
Repeatability and precision. The RSD% of peak areas was used for judgment on the repeatability of the analyte using six different preparations at the same target (500 µg/mL of MP) concentration. It was found to be 1.9% and 0.28% within intra-precision and 1.8% and 0.43% at the inter-precision for the MP and MPHS respectively over 2 days as it demanded in repeatability requirements RSD% < 2.0% 31 as Table 7 manifested.

Robustness.
The results of conscious small changes included a flow rate ± 0.005 mL/min and acetonitrile (± 1%) was determined using RDS%. The RSD% was found to be ≤ 3% in all cases as shown in Tables 8 and 9. It is clear for man there is a reverse proportion between the retention time and the ratio of the organic modifier of the acetonitrile 28 . Where the retention time increases by decreasing the organic ratio and vice versa. This assures the principle chromatographic rule "likes to dissolve likes or likes attract likes" 24-27 . Ruggedness. The results of conscious major and observable changes include analyst-analyst, day-day, and column-column. Data was presented as shown in Tables 10, 11, 12. RSD% was found to be < 3% in all cases 23 .
Specificity and selectivity. The current method supplied us with highly specific data about the resolution and separation performance of the adjacent co-eluted peaks for the MP, 17-MPHS, and MPHS principal peaks. The smallest resolution was found to be 2.54 in the case of MP + MPSS + Buffer as tabulated in Table 13.
Test of the validated method of the local market product of UP Pharma in Egypt. Methylprednisolone 1.0 g vials batch number (221,160) after the constitution stability studies. The tabulated results of the stability studies in Table 14 confirmed the stability and validity of the use of the MP solutions after constitution using WFI, sodium chloride 0.9% wt/v, and glucose 5%wt/v solutions at 5 ± 3 °C for 24 h. Where the assay % was found to be within the acceptance criteria (90-110%) of the stated amount and did not exceed 2.0% from the starting assay at zero time. Also, the results manifested that the method did not affect the composition of the different initiators of the solvent on the retention time over the study.

Conclusions
The validated method was evaluated and it was found to be sensitive to detecting the low concentration of the free Methyl Prednisolone and Methyl Prednisolone hemi succinate at LOD 143.97 ng/mL and 4.49 µg/mL respectively with LOQ 436.27 ng/mL and 13.61 µg/mL. Also, the method was found to be accurate from concentration level 70 µg/mL to 120 µg/mL with high accuracy for free Methyl Prednisolone and Methyl Prednisolone hemi succinate (98.8-99.4%) and (99.4-99.9%) respectively, precise and repeatable over two days with intra precision and inter precision. The linearity of the method was conducted in the range 250 µg/mL to 750 µg/mL with excellent regression coefficient R 2 = 0.9998-0.99999 for free Methyl Prednisolone and Methyl Prednisolone hemi succinate respectively. The method's robustness was evaluated through minor deliberated changes in implementation as different flow rates, different mobile phase compositions, different days, and analysts. It proved its high capability to achieve the requirement of the chromatographic system suitability as the following, theoretical plates and column efficiency ≥ 2000, USP tailing at ≤ 2.0. Finally, the selectivity and specificity of the current method were confirmed by realizing the minimum resolution between the Methyl Prednisolone principal peak and the most adjacent related impurity peak at 2.54. The validated method proved its performance capability in the separation of the Methyl Prednisolone principal peak from any other appearance-forced degradation peaks. Chemical structure Figure  www.nature.com/scientificreports/ www.nature.com/scientificreports/  Table 7. Repeatability of MP and MPHS. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/  Table 11. Column-column effect on MP and MPHS.